International Publication No. WO 1997/019054 and corresponding U.S. Pat. Nos. 6,083,988; 6,197,825; and, 6,291,702 disclose certain chromotropic nitrone radical scavenging agents, methods for making these agents, and methods for their use. These compounds are effective in trapping free radicals, and thus have utility as antioxidants in biological systems. Additional information on these and similar compounds was published in Becker et al., J. Am. Chem. Soc., 124:4678-84 (2002).
Radical scavenging is an important method for garnering information on free radicals that are difficult to impossible to detect by direct spectroscopic observation because of their exceedingly short lifetimes and low concentrations. Two classes of radical scavenging agents that have received the most attention are nitroso compounds and nitrones. Of these, nitrones have been used more frequently, especially in biological systems.
The most commonly cited drawbacks of spin trapping agents (or radical scavengers) bearing a nitroso functionality are instability and toxicity. Because of these undesirable characteristics, researchers often use nitrone spin traps despite the fact that their nitroxide spin adducts generally provide less structural information from electron spin resonance (ESR) than adducts from nitroso based spin traps. Furthermore, due primarily to disproportionation, nitroxides obtained from the addition of certain carbon-centered radicals (tertiary alkyl and aryl) to the most widely used nitrone spin traps (alpha-phenyl-N-tert-butylnitrone (PBN), pyridine N-oxide-4-N-tert-butylnitrone (POBN) and dimethylpyrroline N-oxide (DMPO)) are less persistent than those obtained from addition of such radicals to nitroso compounds.
The use of isotopically labeled spin traps or the application of special equipment consisting of GC/MS or HPLC-interfaced ESR spectrometers designed to detect, isolate, and characterize free radical adducts of nitrone spin traps in biological systems have been reported with varied success.
Nitrones behave as spin trapping agents when a diamagnetic nitrone compound (the “spin trap” or “radical scavenger”) reacts with a transient free radical species (having the “spin”) to provide a relatively more stable radical species (referred to as the “spin adduct”). The spin adduct can be detected by electron paramagnetic resonance (EPR) spectroscopy if the spin adduct has a reasonable lifetime. Thus, information about the spin can be gleaned from a study of the structure and spectroscopic characteristics of the spin adduct.
Various examples of medical applications of radical scavengers are described below.
The toxicity of synthetic β-amyloid peptide preparations toward glutamine synthetase could be correlated with the characteristics of the EPR signal generated by the spin adduct formed from each batch of synthetic β-amyloid peptide and the spin trap PBN. See, Hensley, et al., NeuroReport 6:489-492 (1995). β-Amyloid peptides are neurotoxic substances that are postulated to be involved in the etiology of Alzheimer's disease.
Low molecular weight nitroxides are non-immunogenic. Moreover, they are typically cell permeable and can exist as a non-toxic, stable free radical capable of partitioning among various cellular compartments. Being paramagnetic, nitroxides are detectable by EPR spectrometry and may serve as contrast agents in magnetic resonance imaging (MRI). See, Brasch, Radiology 147:781 (1983); Keana, et al., Physiol. Chem. Phys. Med. NMR 16:477 (1984). Nitroxides have also been used as biophysical markers to probe cellular metabolism, oxygen level, intracellular pH, protein/lipid mobility and membrane structure. Hence, nitroxides find use in a number of diagnostic methods to determine the physiological/medical condition of a subject or the biophysical characteristics of a given sample, including samples obtained from a biological fluid.
Free radicals and oxidative damage have been implicated in brain aging and several neurodegenerative diseases. See, Socci, et al., Brain Research 693(1-2):88-91 (1995). Chronic treatment of aged rats with certain compounds, including the spin trapping agent alpha-phenyl N-tert-butylnitrone (PBN) and the antioxidant alpha-tocopherol (vitamin E), was found to benefit (i.e., improve) age-related changes in cognitive performance.
In vitro and in vivo evidence is mounting that the administration of antioxidants can strongly reduce the rate of progression of lesion formation associated with the process of atherosclerosis. Based on several experimental models, including low density lipoprotein (LDL)-receptor-deficient rabbits, cholesterol-fed rabbits and cholesterol-fed non-human primates, several antioxidants have manifested a 50-80% reduction in the rate of progression of lesions. The effectiveness of probucol, butylated hydroxytoluene (BHT), N,N′-diphenylphenylenediamine, and vitamin E are attributed to their respective antioxidant potentials and to the proposition that oxidative modification of LDL contributes to the progression of atherosclerosis. See, Steinberg, Lancet 346(8966):36-38 (1995). The one-electron oxidative potentials (vs. NHE) of vitamin E in an aqueous solution at pH 7 and 20° C. is 0.48 V. The oxidative potentials of PBN, POBN, and DMPO range from about 1.5-2.0 V.
Further, Downs, et al., Int'l J. Immunopharmacol. 17(7):571-580 (1995), have shown that a cyclic nitrone antioxidant, MDL 101,002, reduces organ dysfunction and cytokine secretion induced by lipopolysaccharide (LPS) administered to rats. The ability of MDL 101,002 to prevent LPS-induced pulmonary edema, leukopenia and thrombocytopenia was also tested. It was found that MDL 101,002 prevented pulmonary edema and partially reduced thrombocytopenia, but failed to prevent leukopenia. These results were consistent with the role that oxygen free radicals played in the development of endotoxin-induced organ dysfunction and shock. It was suggested that free radical scavengers could reduce the mortality consequent to sepsis by organ dysfunction, at least in part, through a reduction in free radical-stimulated cytokine secretion.
Allergic reactions generate reactive oxygen species, including superoxide anions, which usher the influx of inflammatory cells to the site of allergen challenge and contribute to allergic inflammation. The inflammation may, in turn, lead to cell or tissue injury. For allergic reactions in the lung, these processes are also accompanied by increased vascular permeability and changes in airway mechanics. See, Sanders, et al. Am. J. Respir. Crit. Care Med. 151:1725-1733 (1995). Thus, the administration of radical scavenging agents to the site of challenge may reduce the inflammatory response and help reduce tissue or cell damage.
Separately, oxygen-derived free radicals are suspected in playing a role in cytotoxicity during episodes of allograft rejection/destruction following infiltration of the graft by mononuclear cells. The administration of radical scavengers thus may inhibit or reduce the incidence of allograft rejection. See, Roza, et al., Transplantation Proceedings 26(2):544-545 (1994).
New reagents that could visually signal the formation of oxidative species would be extremely useful not only in skin tests or in cell culture, but also in determining, for example, the compatibility of a patient's white blood cells with a particular kidney dialysis membrane. In vitro colorimetric assays would be of great utility.
PBN has been shown to offer protection in the cardiovascular disease area, in particular, by trapping free radicals generated during ischemia-reperfision-mediated injury to the heart. See, e.g., Bolli, et al. J. Clin. Invest. 82:476 (1988). The benefits of trapping free radicals generated in similar types of injury to the brain of experimental animals has also been demonstrated. See, e.g., Oliver, et al. Proc. Nat'l. Acad. Sci. USA 87:5144 (1990); Carney, et al. Proc. Nat'l. Acad. Sci. USA. 88:3636 (1991); Floyd. Science 254:1597 (1991). Oxidative damage to protein and DNA is mediated by oxygen free radical intermediates, leading to strand breaks and base modifications. Enzymes, such as glutamine synthetase, can also be inactivated by oxidative processes. Such damage can be observed, for example, in animals subjected to brain ischemia/reperfusion injury. See, Floyd, et al. Ann. Neurol. 32:S22-S27 (1992).
Evidence is also available that PBN inhibits oxidative modification of cholesterol and triglycerides of low density lipoproteins (LDL). Oxidative modification of LDL, along with lipid peroxidation and free-radical mediated reactions, is a process that is implicated in the initiation of atherosclerosis. See, e.g., Steinberg, et al. N. Engl. J. Med. 320:915 (1989); Esterbauer, et al. Ann. N.Y. Acad. Sci. 570:254 (1989).
Free radicals and oxidative damage have been proposed as the underlying reasons for aging, chronic and degenerative diseases of aging, and acute clinical conditions. Daily administration by intraperitoneal injection of PBN to an aged animal model showed that PBN offered a remarkable extension of the lifespan in both male and female populations. See, Packer, et al., Biochem. Biophys. Res. Commun. 211(3):847-849 (1995). PBN could have prophylatic value against the onset of, at least, pathological senescence.
Ames and co-workers (Proc. Nat'l. Acad. Sci. USA 92:4337-4341 (1995)), hypothesized that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells. It was found that senescent cells, i.e., those cells that have ceased growth in culture after a finite number of population doublings, excise from DNA four times more 8-oxoguanine per day than early-passage young cells. Also, levels of 8-oxo-2′-deoxyguanosine in DNA of senescent cells are about one third higher than those found in DNA of young cells. Most interestingly, PBN effectively delayed the onset of senescence and rejuvenated near senescent cells, perhaps acting as either an antioxidant or as a radical scavenging agent.
There are several non-medical applications for the use of radical scavengers, such as nitrones. A number of factors influence fat stability and the formation of lipid oxidation products. Increased unsaturation, increased frying time, increased exposure of the oil to air, and increased trace metal content will all result in decreased oxidative stability. The presence of silicones in a frying oil will cause increased oil stability by yet unknown mechanisms. Published data indicates that filtration of oils through certain active adsorbents will increase the useful frying life of an oil during actual fryer use by removal of colored materials, free fatty acids and other oxidation products.
Usually peroxides decompose at about 150° C. Therefore at frying temperatures, the accumulation of peroxides does not occur. Peroxide values usually are a measure of lipid oxidation at lower temperatures such as those used for storage of fats or a product. The relationship between storage time and peroxide value can then be used to measure quality.
The Schall oven test involves simply putting a small amount of the fat into a beaker and placing it into an oven under standardized conditions at 60° C. to oxidize the sample. Samples then are taken and peroxide values determined. There are many other tests available to check frying oil quality, all which purport to inform the operator when to do something with the used fat—either filter it through active filters, discard it, or dilute it with a less degraded fat. Some tests which have been used to check frying oil quality are the saponification color index, 2,6-dichloroindole phenol color test, methylene blue color test, and iodine color scale. These tests allegedly determine when the fat has degraded and can no longer produce a high quality food product. For instance, the Rau test from E. Merck is a colormetric test kit which contains redox indicators that react with total oxidized compounds in a sample. It has a four color scale and is used for diagnoses of fat quality. The fourth color scale indicates a degraded oil and the oil should be discarded. All these tests differ in reliability and may be more tedious to perform than necessary.
Surprising difficulty in starting a lawn mower, trail bike, outboard motor, or similar infrequently used gasoline engine, is caused by “bad” petroleum. Petroleum is subject to autoxidation, like oils in foods and in the human body. When gasoline is left for any long period (e.g., a few months or more), gums are formed by the reaction of oxygen with unsaturated components of the fuel. BHT (also known as 2,6-di-tert-butyl p-cresol) is a U.S. government approved gasoline additive that meets military requirements for gasoline stability. A half pound of BHT added to 1,100 gallons of gasoline prevents gum formation when gasoline was stored in sealed (with standard rubber washers) 5-gallon cans for periods up to two years in the Mojave desert in fill sunlight, compared to a storage life of only a few months for unprotected gasoline. The amount currently recommended for military use is 1 pound BHT to 1,100 gallons of gasoline. For even longer storage, BHT, alone, may not be sufficient to prevent spoiling of the fuel.
Other materials that are affected by similar aging mechanisms include plastics, rubber, paint asphalt, roofing shingles, oils and lubricants.
Accordingly, radical scavengers exhibit a wide range of properties that are applicable to many end uses. There exists a continuing need to discover new, effective substances exhibiting free radical/spin trapping and/or antioxidant activity which are potentially useful for a wide range of analytical preservative, diagnostic, prophylactic and therapeutic applications.